Intracellular compartmentation has been proposed as an important determinant in the regulation of metabolic processes. Recent observations suggest that "free" heme, individual hemoproteins, bilirubin formed in the liver from these sources of heme, and bilirubin in transit through the liver from plasma to bile, exist in structural and/or functional subcellular compartments in hepatic parenchymal cells. The present proposal describes several approaches designed to study this largely unexplored area of liver function. Attempts will be made to define the metabolic pathways of bile pigment derived from both intra-and extrahepatic heme sources, and to characterize the extent and metabolic significance of heme and bilirubin compartmentation in the liver. Recently developed probes of heme and bile pigment metabolism (i.e., isotopically labeled precursors of heme and bilirubin) will be applied in a variety of experimental systems, including intact rats, the isolated perfused rat liver and adult rat hepatocyte monolayer culture. Specifically, attempts will be made to determine whether heme (e.g., hemoglobin) taken up by the hepatocytes from plasma is utilized in the synthesis of hepatic hemoproteins (e.g., cytochromes and catalase). The rate of 14CO production from prelabeled hemoproteins will be used to assess the relative contributions of individual hemoproteins to the overall formation of hepatic bilirubin. A kinetic model will be formulated to describe the functional compartments and transport channels for bilirubin formed from both extrahepatic and intrahepatic sources. The subcellular localization of individual heme and bilirubin compartments will be examined using electron autoradiography and subcellular fractionation of hepatocytes. It is anticipated that the information obtained in the course of these studies will contribute to an improved understanding of the regulation of heme and bilirubin metabolism, and have a direct bearing on the hepatic transport of xenobiotics and on clinical disorders, such as the hepatic porphyrias and various types of jaundice.